Resveratrol promotes liver cell survival in mice liver-induced ischemia-reperfusion through unfolded protein response: a possible approach in liver transplantation

Background Ischemia-reperfusion (I/R) of the liver is a multifactorial condition that happens during transplantation and surgery. The deleterious effects of I/R result from the acute production of reactive oxygen species (ROS), which can trigger immediate tissue damage and induce a series of destructive cellular responses, including apoptosis organ failure and inflammation. The production of ROS in the I/R process can damage the antioxidant system and cause liver damage. Resveratrol has been shown to have antioxidant properties in several investigations. Here, we address the therapeutic effect of resveratrol on I/R-induced liver injury by focusing on unfolded protein response (UPR) signaling pathway. Methods Five minutes before reperfusion, resveratrol was injected into the tail vein of mice. They were ischemic for 1 h and then re-perfused for 3 h before being slaughtered (I/R). The activity of liver enzymes and the expression levels of genes involved in the unfolded protein response pathway were used to measure the hepatic damage. Results Our results revealed that the low dose of resveratrol (0.02 and 0.2 mg/kg) post-ischemic treatment significantly reduced the ALT and AST levels. In addition, compared with the control group, the expression of UPR pathway genes GRP78, PERK, IRE1α, CHOP, and XBP1 was significantly reduced in the resveratrol group. In the mice that received lower doses of resveratrol (0.02 and 0.2 mg/kg), the histopathological changes induced by I/R were significantly improved; however, the highest dose (2 mg/kg) of resveratrol could not significantly protect and solve the I/R damage. Conclusion The findings of this study suggest that hepatic ischemia occurs after liver transplantation and that receiving low-dose resveratrol treatment before reperfusion may promote graft survival through inhibition of UPR arms, especially PERK and IRE1α. Supplementary Information The online version contains supplementary material available at 10.1186/s40360-022-00611-4.

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Totonchi et al. BMC Pharmacology and Toxicology (2022) 23:74 function after liver transplantation (LT) [3]. Because liver cells are the primary source of multiple metabolic pathways and have a high basal-specific metabolic rate, they are more vulnerable to the exacerbating effects of ischemia, such as hypoxia and ATP depletion [4]. The destructive effect of I/R stems from generating reactive oxygen species abruptly after re-oxygenation, damaging tissues directly, and triggering a series of destructive cellular reactions, resulting in apoptosis, inflammation, and organ failure [5]. A deficiency of ATP causes major intracellular organelles to dysfunction and triggers stress responses, e.g., endoplasmic reticulum (ER) stress response [6]. The endoplasmic reticulum plays a role in calcium homeostasis, lipid biosynthesis, and correct protein folding, helping the inhibition of the accumulation of unfolded and misfolded proteins in the ER [7]. Endoplasmic reticulum stress is a condition caused by ER malfunction (ERS). In this circumstance, the proper balance between protein synthesis and protein folding in the ER is broken. Depending on the severity and duration of the stress, several compensatory processes, such as the unfolded protein response (UPR), activate the adaptive defense systems [8,9]. The unfolded protein response (UPR) is a defensive mechanism activated by ERS that inhibits the protein translation, destroys the misfolded proteins, and activates the signaling pathways that boost the synthesis of molecular chaperones involved in protein folding [10]. UPR is activated when misfolded or unfolded proteins accumulate in the endoplasmic reticulum lumen [11,12]. It is worth mentioning that cell death occurs under excessive stress and when the adaptive pathways fail to compensate for the ER normal function [13,14]. In mammals, UPR consists of three specific sensors or arms located on the outer membrane of ER in association with GRP78. The ER stress response is regularly monitored by three specific UPR sensors, including inositol-requiring enzyme 1α (IRE1α) and IRE1β, protein kinase R (PKR)like endoplasmic reticulum kinase (PERK), and activated transcription factor 6 (ATF6; α and β are the same species) [15]. GRP78 (78 kD glucose-regulated protein), also named immunoglobulin binding protein (BiP)-master ER chaperone-binds to the abovementioned sensors and inhibits their activation under normal conditions [16,17]. When unfolded proteins accumulate in the ER, GRP78 dissociates from ATF6, IRE1, and PERK arms. This separation leads to the release of these ER sensors and activates the UPR pathway [18]. In the presence of ER stress, activated PERK dissociates from GRP78 and phosphorylates eukaryotic initiation factor 2 (eIF2) to relieve ER pressure. Dissociated PERK then inhibits the translation of extracytoplasmic mRNA and prevents newly synthesized proteins from entering the ER. Furthermore, activated IRE-1 has an effect on the X-box-binding protein (XBP-1) mRNA transcription factor via endonuclease activity. XBP-1 regulates the expression of several genes involved in the ER quality control, ERAD (ER-related degradation), and apoptosis when it is spliced and activated [19]. Finally, activated ATF6 is disassociated from GRP78 and transported to the Golgi organelle. The transported ATF6 is then activated by site 1 and site 2 (SP) proteases via regulated intramembrane proteolysis (RIP). The activated ATF6 cytoplasmic domain (50 kDa) is translocated to the nucleus and increases the levels of ER chaperones, XBP-1, and protein folding [12,20]. The activation of these arms also contributes to cell growth suppression by inducing C/EBP homologous protein (CHOP), a DNA damage-inducible protein (GADD34), and pro-apoptotic BCL-2 family proteins. Overall, UPR has two faces in various situations based on the cellular nature, intensity, and time to cope with stress; they are cell-protective and cell-cytotoxic roles [21,22]. In the recent decade, researchers have begun to search for new drug discovery and strategies for therapeutic interventions based on endoplasmic reticulum stress. ER stress and UPR are associated with such complications such as inflammation, neurodegenerative diseases, metabolic syndrome, and cancer [9,21]. It has been shown that the ectopic expression of activated ATF6 can reduce the damage to the heart caused by of I/R. ATF6 reduces the I/R damage by regulating the expression of the antioxidant genes including catalase, peroxiredoxin 5 (Prdx5), and Vimp as selenoprotein, especially by reducing the ROS levels [23]. Recent studies have shown that UPR pathways are activated in two phases when livers are reperfused, as during the ischemic phase, IRE-1 is induced and PERK and eIF2α are inhibited before these effects are reversed during reperfusion [24].
Resveratrol (trans-3,49,5-trihydroxystilbene) is a plantderived polyphenolic compound which is found abundantly in various plant species such as grapes that have attracted great interest from researchers due to its anticancer, anti-aging, anti-neurodegenerative, and antiinflammatory properties [25]. To date, several clinical trials have tested the efficacy, safety, and pharmacokinetics of resveratrol in the prevention and treatment of different pathological conditions (www. Clini calTr ials. gov).
Most of these studies have tested resveratrol-mediated effects in central nervous system disorders and metabolic disorders including insulin resistance, dyslipidemia, and non-alcoholic fatty liver disease (NAFLD). It was shown to be involved in the activation of macrophages, T cells, and natural killer cells [26]. All these effects are due to its ability to remove the ROS, inhibit cyclooxygenase (COX), and trigger anti-inflammatory pathways via SIRT1 activation [27]. The Sirtuin 1 (SIRT1) gene encodes a nicotinamide adenosine dinucleotide (NAD)-dependent histone deacetylase that is involved in a variety of biological processes such as longevity, stress response, and cell survival. SIRT1 activated by RSV reduces the expression of inflammatory factors such as TNFa, IL-1b, IL-6, metalloprotease (MMP)-1, MMP-3, and NFkB-mediated Cox-2 [27]. Resveratrol protects the hepatic cells against experimental ischemia-reperfusion injury by suppressing the expression of hypoxia-inducible factor (HIF1α) and vascular endothelial growth factor (VEGF) [28]. Previous reports have demonstrated that resveratrol suppresses the nuclear translocation of NF-κB in the rat hepatocytes (BRL-3A) cells and decreases the TNF-α and IL-1β production in vitro and in vivo [29]. Resveratrol has been shown to have a significant effect on endoplasmic reticulum stress regulation, but this effect is highly complex [30]. RSV plays a role in inducing the stress of the endoplasmic reticulum to promote apoptosis of cancer cells. Other studies have shown that RSV can improve cardio-myocyte hypertrophy by inhibiting ER stress [31]. Moreover, resveratrol can prevent hepatic steatosis and endoplasmic reticulum stress in rats fed with a high-fat diet [32]. SIRT1 deacetylates XBP1 and inhibits its transcriptional activity to promote ER stress-induced apoptosis, and SIRT1 suppresses the PERK-eIF2-dependent translational inhibition in mammals, according to recent findings [12][13][14]. In liver cells, sirtuin 1 (SIRT1) which is a negative regulator of UPR signaling [33] leads to the reduction of ER stress by preventing UPR overactivation, induces ER chaperone expression, and inhibits the proapoptotic activity of UPR targets [34]. Thus, it may mean that SIRT1 can be regulated through specific SIRT1 activators, e.g., resveratrol (3, 5, 4′trihydroxystilbene; RSV), to restore ER homeostasis [35]. Although the antioxidant effect of resveratrol has been evaluated in multiple studies [36][37][38], few studies have demonstrated the effect of resveratrol on the UPR pathway. Therefore, this study aims to evaluate the effects of resveratrol on ischemia/ reperfusion injury and UPR pathways and its specific arms to ultimately improve the success rate of liver transplantation.

Animals
Male BALB/c mice obtained from Shiraz Medical University Transgenic Research Center were used in this study. The mice were kept in the laboratory for 1 week to adapt to the laboratory conditions. The animals were raised, treated, and handled exactly based on the ethical treatment and animal handling program of the Institutional Ethics Committee and Research Advisory Committee of Shiraz University of Medical Sciences (IR. SBMU.MSP.REC.1397.836).

Ischemia-reperfusion (I/R)
The mice were anesthetized by IP (intraperitoneal) injection of 100 mg/kg ketamine and 10 mg/kg xylazine [39]. After a midline abdominal incision, partial ischemia was achieved by clamping the portal vein and hepatic artery supplying the middle and left lobes (about 70% of the liver parenchyma) for 1 h. Occlusion of these vein and artery caused an immediate change in liver color to a lighter hue. The clamp was then removed to allow the tissue to be re-perfused. Next, the skin and muscle tissue were stitched together during the reperfusion process. The animals were sacrificed 3 h after reperfusion [40]. The left and middle lobes of the liver were removed and stored at −80°C.

Principal materials
Rabbit and mouse primary antibodies against human, namely GRP78 and IRE1α, were provided by the Cell Signaling Technology, Co. (Beverly, MA, USA). Resveratrol (Cas Number: 501-36-0) was purchased from Sigma-Aldrich Co. (Oakville, ON, Canada). GAPDH was prepared from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and anti-rabbit IgG (whole molecule) and anti-mouse immunoglobulin G (IgG) (Fab specific) as secondary antibodies were provided by Sigma-Aldrich (Oakville, ON, Canada). The bicinchoninic acid (BCA) protein assay kit was provided by Thermo Fisher Scientific (Winnipeg, MB, Canada).

Administration of resveratrol
Dimethyl sulfoxide (DMSO) was used to dissolve resveratrol [41] in different concentrations (0.02, 0.2, 2.0 mg/kg) [40] and injected into the mouse tail vein 5 min before reperfusion. The mice underwent 1 h of ischemia and were sacrificed after 3 h of reperfusion (I/R). Afterwards, the mice were randomly divided into 5 separated groups (Table 1). We put 5 mice in each group and selected the minimum number of mice based on "resource equation method" and the formula (E=total number of animalstotal number of groups) [42].

Blood sampling
Blood samples were collected from the heart 3 h after reperfusion. The samples were centrifuged (3500 rpm, 4°C for 10 min), and the plasma was kept at −20°C.
Evaluation of liver damage was done by measuring the activity of cytolytic enzymes in plasma-aspartate aminotransferase (AST) and alanine transaminase (ALT) using diagnostic colorimetric kits (BioSystem, Spain) by prestige instrument (Hitachi Japan).

Total RNA isolation and real-time PCR analysis
According to the manufacturer's protocol, the total RNA was extracted from the liver using RNAiso Plus reagent (TaKaRa Biotechnology, Dalian, Liaoning, China) and stored at −75°C. We used BioFact cDNA synthesis kit (Biofact, Daejeon, Korea) to synthesize cDNA. Table 2 provides specific primer sets for target genes. The expression levels of GRP78, PERK, ATF6α, CHOP, and XBP1 were performed by applying SYBR Green Master Mix on a RotorGene Q real-time PCR machine (Qiagen, Hamburg, Germany). In short, a total volume of 20 μl in a 0.2ml test tube was run at 95°C for 30s, and then, 40 cycles were performed at 95°C for 20s and 60°C for 40 s. Each experiment was repeated 3 times. The expression levels of GRP78, PERK, ATF6α, CHOP, and XBP1 were standardized to the GAPDH genes as housekeeping genes by applying the 2-ΔΔCt method.

Western blot assay
Frozen liver samples were homogenized in a lysis buffer in the presence of proteinase inhibitors to isolate total liver protein. Total protein was extracted and lysed by applying NP40 Lysis Buffer [30]. The bicinchoninic acid method was used to quantify the collected protein. Depending on the type of protein, 20 to 40μg of total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and electroblotted onto a 0.2-μm nitrocellulose membrane. Subsequently, the membrane was blocked by applying 5% free milk and incubated with primary antibodies (IRE1α, GRP78, GAPDH) overnight at 4°C. Subsequently, the membranes were treated for 60 min at 25°C with horseradish peroxidase (HRP)-conjugated secondary antibody. Finally, protein bands were observed by incubating the membrane with enhanced chemiluminescence (ECL) reagents and detected using the ChemiDoc ™ MP imaging system (BioRad, USA). The intensity of the band was quantified using Image lab software, and all blots were standardized to GAPDH as an internal control protein.

Histological analyses
For histopathological evaluation, liver sections were fixed with formalin (10%) and then dehydrated with ethanol. In the next step, the fixed sample was embedded in paraffin and cut into 5-μm wide sections. Tissue samples were de-paraffinized in xylene, and finally, hematoxylin-eosin (H&E) staining was used to assess hepatocyte vacuolation, sinusoidal congestion, and focal parenchyma inflammation [40]. Histopathological sections were evaluated according to Suzuki's histologic grading [43]. The degree of cytoplasmic vacuolization, sinusoidal congestion, and parenchymal necrosis was scored from 0 to 4 as previously described [44]: 0=none, 1=mild, 2=moderate, 3=marked, and 4=severe to diffuse.

Statistics
The results are shown as mean ± SEM. Based on the results of the normal test, a parametric test, or a nonparametric test, was applied to compare different groups (control, DMSO, RES 0.02, RES 0.2, and RES 2). One-way ANOVA was applied to compare the variables between the treated and control groups. Tukey's post hoc test was applied for parametric test, and Kruskal-Wallis post hoc was used for non-parametric analysis. On the other Table 2 The sequence of primer sets applied for real-time PCR assays

Effect of resveratrol on liver injury caused by hepatic ischemia-reperfusion
The release of ALT and AST into plasma reflects the hepatic injury. Three hours after reperfusion, evaluation of plasma levels of ALT and AST revealed that there was no significant association between I/R+Res 2 group and I/R + DMSO and I/R groups. However, post-ischemic treatment with low doses of resveratrol (0.02 and 0.2 mg/ kg) significantly decreased the levels of ALT and AST compared to I/R, I/R + DMSO, and I/R+Res 2 groups (Fig. 1).

Effect of resveratrol on the mRNA expression levels of GRP78, PERK, ATF6α, CHOP, and XBP1 in ischemia-reperfusion injury
For the evaluation of the effect of different doses of resveratrol on the genes involved in the UPR pathway, realtime PCR was used. Gene expression of GRP78, PERK, CHOP, and XBP1 was significantly decreased in the resveratrol groups (0.02, 0.2, and 2 mg/kg) compared with the IR and IR+DMSO groups (p<0.01). In addition, the effect of resveratrol at a lower dose was significantly greater than at a higher dose. In fact, expression of GRP78, PERK, CHOP, and XBP1 was significantly higher in the group receiving resveratrol at a dose of 2 mg/kg (Res 2mg/kg) compared to the other treatment groups (Res 0.2mg/kg and Res 0.02mg/kg) (p<0.05) ( Fig. 2A-E). However, none of the treated doses of resveratrol had any effect on ATF6α expression in the treatment groups, and a comparison between different treatment groups and control groups did not reach a statistically significant threshold (Fig. 2B).

Effect of resveratrol on the protein level of IRE1α and GRP78
After ischemia-reperfusion, the protein levels of IRE1α and GRP78, as a master regulator of the UPR pathway, were determined by immunoblotting (Fig. 3A). The results of immunoblotting revealed that resveratrol at all three doses (0.02, 0.2, and 2 mg/kg) significantly reduced IRE1α protein levels compared to IR and IR+DMSO groups (Fig. 3B). Moreover, in line with the real-time PCR results, resveratrol at lower doses (0.02 and 0.2 mg/ kg) significantly decreased the protein level of GRP78 (Fig. 3C). However, a high dose (2 mg/kg) of resveratrol had no significant effect on the protein level of GRP78 compared to IR and IR+DMSO groups. There was no Fig. 1 The effect of resveratrol on the serum ALT and AST levels after I/R. Resveratrol was injected into the tail vein of the mouse 5 min before reperfusion. The mice underwent 1 h of ischemia and were sacrificed after 3 h of reperfusion (I/R). Data are expressed as mean ± SD. According to the Tukey post hoc test used for the comparison between groups, the groups with the same superscript letters did not have significant differences when α = 0.05 (p ≥ .05). Thus, various letters show considerable differences (p <0.05) significant difference between IRE1α and GRP78 expression levels in the I/R group compared to I/R +DMSO groups.

Histopathological analysis
After 3 h of reperfusion, the animals were sacrificed, and liver specimens were sent for pathological evaluation. After ischemic perfusion, the main histological findings were sinusoidal congestion, vacuolation of hepatocytes, and focal parenchyma inflammation (Fig. 4A, B, and F). In addition, in the IR and IR+DMSO groups, the infiltration of inflammation and inflammatory cells into the sinusoids was obvious. Severe hepatocyte vacuolation, sinusoidal congestion, and focal parenchyma inflammation were observed in the IR group (Fig. 4A). In the group which received lower doses of resveratrol (0.02 and 0.2 mg/kg), these histopathological changes were significantly improved (Fig. 4C, D, and F) (p <0.05). However, higher doses (2 mg/kg) of resveratrol did not have a significant protective effect in solving the I/R injury of the treated mice (Fig. 4E). As depicted in Fig. 4F, low doses of resveratrol (0.02 and 0.2 mg/kg) significantly decreased the focal inflammation compared to 2 mg/kg of resveratrol. It has been shown that 0.2 mg/kg resveratrol declined the number of Kupffer cells compared to the dose of 2 mg/ kg. The score of histopathological changes of sinusoidal congestion, vacuolation of hepatocytes, focal inflammation, and the number of Kupffer cells was shown in Fig. 4G.

Discussion
In this study, we assessed the effect of resveratrol on ischemia/reperfusion injury and UPR pathway in the in vivo system. Considering the cross-talk between oxidative stress, ER stress, and UPR arms, we evaluated the effect of resveratrol on UPR sensors to regulate the cellular antioxidant capacity. The goal of this project was to evaluate for the molecular mechanism of resveratrol and answer the following novel questions: (1) is the UPR process involved in the effects of resveratrol on the I/R injury of the liver? (2) Which arm of the UPR is mostly involved in the effectiveness of resveratrol on damaged liver? The results of the current study suggest that post-ischemic treatment with resveratrol can increase the survival of liver allografts after transplantation at lower doses. Post-ischemic treatment with low doses of resveratrol significantly decreased the levels of ALT and AST. In addition, a significant reduction in the expression of GRP78, Perk, IRE1, CHOP, and XBP1 was identified compared to the IR+DMSO group. However, the effects of different doses of resveratrol on the expression of the ATF6 gene did not reach a statically significant threshold. Ischemia-reperfusion injury (I/R) is a multifactorial phenomenon which occurs during transplantation events and often impairs the transplant function early after liver transplantation [45]. The mechanism of injury is closely related to the inflammatory response, which leads to microcirculation failure, followed by necrosis and cell death [45]. Although many studies have been done on I/R injury of the liver, the exact mechanism that causes transplant rejection is not fully understood. Due to the high level of metabolism, liver cells are highly susceptible to the destructive effects of I/R, including reduced levels of ATP and hypoxia, which can subsequently cause damage to various organs, such as the endoplasmic reticulum [46]. Liver cells respond to endoplasmic reticulum stress through a defense process called the unfolded protein response (UPR) [46].
Resveratrol is a polyphenolic compound that has a protective effect on the liver cells and can improve the survival rate of liver allografts after transplantation [47]. Resveratrol can reportedly be used as a signal molecule in the tissues and cells to regulate the gene and protein expression. Stimulation of these proteins and enzymes may explain some of the antioxidant properties in cells [48]. Genetic regulation is sufficient to explain some of the cytoprotective effects of resveratrol, as well as its effects on blood flow, cell death, and the inflammatory cascade [49]. In recent years, the therapeutic effect of resveratrol on liver diseases has been confirmed in several studies. Resveratrol significantly improves the survival rate after liver transplantation and reduces apoptosis, necrosis, and ischemia-induced fat deposition in animal models [50].
In 2005, Sylvain et al. demonstrated for the first time that resveratrol could be used as a signal molecule in cells to regulate the expression of genes and proteins. The stimulation of these proteins and enzymes can explain some of the antioxidant properties in cells [51]. Consistent with our study, Khabbar et al. demonstrated that low-dose resveratrol helped resolve liver inflammation secondary to liver surgery and/or liver allograft rejection [40]. Therefore, they concluded that trans-resveratrol might be a viable treatment option that can selectively inhibit the self-cutting cycle of inflammatory damage [40].
A number of studies have shown that resveratrol is a new type of anti-inflammatory agent, which can improve the endoplasmic reticulum environment by reducing the protein load on the endoplasmic reticulum and maintaining the integrity of the ER membrane [52]. Resveratrol inhibits the expression of genes expressed by the UPR cell response. In this study, it was found that the expression levels of CHOP and GRP78 in low-dose resveratrol were significantly reduced compared to I/R group. With a decrease in the expression levels of GRP78 and CHOP, it can be concluded that the resveratrol induces survival branch of the UPR.
Consistent with this finding, Tabata et al. showed that Vaticanol B (a resveratrol tetramer) prevented the induction of the UPR pathway by inhibiting the expression of CHOP and GRP78 [52]. In fact, ischemia-reperfusion injury leads to an increase in IRE1 and PERK, which in turn leads to cell death by increasing CHOP [53]. Resveratrol regulates these two arms to prevent the cell death. Furthermore, polycystic ovary syndrome (PCOS) patients have been reported to have reduced XBP-1 mRNA expression after resveratrol treatment, which is in line with our findings [54]. Our data also showed that resveratrol reduced the level of XBP-1 expression, while ATF6 expression was slightly decreased or unchanged. In fact, resveratrol can reduce the activity of the ATF6 arm in the UPR pathway, but this reduction was not significant in our study. One of the main reasons for this situation may be related to the interaction between ATF6 and catalase. ATF6 is one of the main inducers of catalase, and complete inhibition of this transcription factor will lead to the loss of antioxidant properties in the cell and ultimately cell death [55].
It can be understood that resveratrol reduces the number of UPR target genes by reducing XBP-1 and simultaneously increases the transcription of inhibitory proteins in the inflammatory pathway by increasing ATF6.
In 2019, Zhao et al. studied the effects of resveratrol on hepatic endoplasmic reticulum (ERS) stress and insulin sensitivity in vivo and in vitro [56]. They showed that resveratrol could reduce liver ERS, thereby improving insulin sensitivity and glucose levels. However, high doses of resveratrol can have damaging effects on the cells, increasing ERS and insulin resistance [56]. It is worth mentioning that resveratrol has yet to be fully examined, according to various research. The in vivo toxicity of resveratrol in rodents has been studied in several publications. It has been observed that a high dose of resveratrol produces pro-oxidant effects in mice via suppressing cyclooxygenase (COX1) expression and prostaglandin (PG2) synthesis, leading to ulcerative damage [57,58]. According to another study, high doses of resveratrol produce renal fibrosis, severe nephropathy, and considerable increases in BUN and creatinine, and their anti-fibrotic benefits are entirely reversed [59]. Only low doses (0.02, 0.2 mg/kg) of trans-resveratrol (penis vein injection) in rats can reduce the liver injury (decrease ALT, AST by roughly 40%) caused by I/R. However, lowdose resveratrol improves the antioxidant defense system and increases the activity of hepatic GPx, Cu/Zn SOD, catalase enzymes, as well as the total and reduced glutathione if all of these effects are completely reversed at high doses of resveratrol (2.0, 20 mg/kg) [40]. Consistent with this finding, our results also showed that low-dose resveratrol (0.02 and 0.2 mg/kg) had a significant protective effect (ALT, AST) on the resolution of I/R damage. In fact, based on several previous published data, it appears that higher doses of resveratrol resemble more to a prooxidant than to an antioxidant and can cause cell damage [40,60]. As there is no more research on the appropriate dosage of resveratrol, more research is needed to confirm our results. Despite numerous intriguing findings, the current study has some limitations. For example, it is much better to analyze all UPR elements on gene and protein expression in order to arrive at a more precise conclusion. According to several related publications, no significant changes were seen in the sham-operated control group without ischemia-reperfusion in liver function tests, histopathological studies, inflammatory factors, as well UPR elements (ATF4, CHOP), and apoptosis factors after (1, 3, 6, 12, and 24 h) post-liver reperfusion [61][62][63][64]. But with these details in our study, considering one group as a sham-operated control is better to make the findings more objective and analyze the influence of the operation on variables as a baseline, particularly in UPR molecular pathways. These points will undoubtedly be taken into account in future research.

Conclusion
Taking into account the effects of the UPR pathway and the cross-talk between oxidative stress, ER stress, and UPR arms in restoring cellular homeostasis, we investigated the ability of resveratrol to regulate cellular antioxidant capacity in this study. According to our findings, resveratrol can reduce the severity of ERS and suppress the expression of UPR arms (PERK and IRE1α) activated by hepatic ischemia/reperfusion in response to ERS. The findings of this study suggest that hepatic ischemia occurs after liver transplantation and that treating recipients with low-dose resveratrol before reperfusion may improve the graft survival by inhibiting UPR arms in particular (IRE1α and PERK). More research is needed, however, to determine an effective and safe dose of resveratrol.
Additional file 1: Figure 1S. The effect of resveratrol on the serum ALT and AST levels after I/R. Resveratrol was injected into the tail vein of the mouse 5 minutes before reperfusion. The mice's underwent 1 hour of ischemia and were sacrificed after 3 hours of reperfusion (I/R). Data are expressed as mean ± SD. According to the Tukey post-hoc test used for the comparison between groups, the groups with the same superscript letters did not have significant differences when α = 0.05 (p ≥ .05). Thus, various letters show considerable differences (p <0.05). Figure 2S. The impact of resveratrol on the expression levels of GRP78, PERK, ATF6α, CHOP and XBP1 after I/R. Sham-operated group, I/R, DMSO, 0.02, 0.2 and 2. The mice's were sacrificed after 1 hour of ischemia and reperfusion (I/R) for 3 hours. Data are expressed as mean ± SD. One-way analysis of variance is used to compare the groups with the same superscript letters, which are not significantly different when α = 0.05 (p ≥ 0.05). However, various letters show considerable differences (p <0.05). Figure 4S. Evaluation of the effect of resveratrol on the liver injury after I/R by histopathological analysis. Sham-operated group with normal liver architecture (A). In mice's in the IR and IR + DMSO groups (B and C), obvious sinusoidal congestion, vacuolation of the hepatocytes (thin arrow), and focal parenchyma inflammation (thick arrow) were obvious. Mice's with mild sinusoidal dilation received resveratrol at 0.02 and 0.2 mg/kg. Abnormal histopathological changes in the resveratrol group (D and E) were significantly improved. A higher dose (2 mg / kg) of resveratrol did not have a significant protective effect on the treatment of I / R injury in mice's (F).


Systematic Tracing of Susceptible Animals to SARS-CoV-2 by a Bioinformatics Framework

Since the outbreak of SARS-CoV-2 in 2019, the Chinese horseshoe bats were considered as a potential original host of SARS-CoV-2. In addition, cats, tigers, lions, mints, and ferrets were naturally or experimentally infected with SARS-CoV-2. For the surveillance and control of this highly infectious disease, it is critical to trace susceptible animals and predict the consequence of potential mutations at the binding region of viral spike protein and host ACE2 protein. This study proposed a novel bioinformatics framework to systematically trace susceptible animals to SARS-CoV-2 and predict the binding affinity between susceptible animals’ mutated/un-mutated ACE2 receptors. As a result, we identified a few animals posing a potential risk of infection with SARS-CoV-2 using the docking analysis of ACE2 protein and viral spike protein. The binding affinity of some of these species is weaker than that of humans but more potent than that of Chinese horseshoe bats. We also found that a few point mutations in human ACE2 protein or viral spike protein could significantly enhance their binding affinity, posing an enormous potential threat to public health. The ancestors of the Omicron may evolve rapidly through the accumulation of mutations in infecting the host and jumped into human beings. These findings indicate that if the epidemic expands, there may be a human-animal-human transmission route, which will increase the difficulty of disease prevention and control.

INTRODUCTION
In December 2019, Coronavirus disease 2019  was first reported in Wuhan, China. The etiological agent of COVID-19 has been confirmed as a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Gorbalenya et al., 2020). COVID-19 rapidly spread and caused a global pandemic. By 22 September 2021, SARS-CoV-2 resulted in 229,543,672 human infections with 4,708,355 fatalities 1 . The virus and its variants continue to circulate globally, posing a serious threat to public health.
To date, seven coronaviruses are known to infect humans, of which HCoV-229E and HCoV-NL63 are alpha coronaviruses, while HCoV-OC43, HCoV-HKU1, SARS-CoV-1, Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 belong to beta coronaviruses. SARS-CoV-2 shares 80% identity with SARS-CoV-1 at the nucleic acid level. SARS-CoV-2 shares 96% nucleic acid similarity with two bats β-coronaviruses, indicating that SARS-CoV-2 may be derived from bat coronavirus . Previous studies have suggested that bats are one of the major natural hosts for coronaviruses such as SARS-CoV-1 and MERS-CoV and are associated with several coronaviruses causing severe human diseases. SARS-CoV-1 was confirmed to derive from bat-origin coronaviruses (Li et al., 2005;Shi and Hu, 2008), and paguma larvata acted as an intermediate host during the transmission of SARS-CoV-1 to humans (Hu et al., 2017). Similarly, MERS-CoV was confirmed to derive from camel-origin coronaviruses, and dromedary camels play an important role in spreading and transmitting viruses (Alagaili et al., 2014;Meyer et al., 2014;Muller et al., 2014;Sabir et al., 2016). Thus, it is critical to reveal susceptible animals, which will favor the origin tracing, prevention, and control of SARS-CoV-2.
Besides human beings, SARS-CoV-2 can infect other mammalians, such as ferrets, cats, dogs, tigers, lions, and mink. A total of 14.7% (15/102) sera collected from cats in Wuhan from January to March 2020 were confirmed positive to SARS-CoV-2 . SARS-CoV-2 can effectively replicate in cats and transmit to uninfected ones through the respiratory droplets. However, other companion pets (dogs) are low susceptible to SARS-CoV-2. SARS-CoV-2 can effectively replicate in the upper respiratory tract of ferrets and does not cause severe symptoms or fatality (Shi et al., 2020). Tigers and lions in Bronx zoo of Wildlife Conservation Society with coughing symptoms were confirmed positive to the RNA of SARS-CoV-2, and these indicated that SARS-CoV-2 could infect tigers and lions (McAloose et al., 2020). In addition, minks were confirmed to be positive for SARS-CoV-2 and died of acute interstitial pneumonia in four mink farms in Netherland during 19-20 April 2020, suggesting that SARS-CoV-2 can infect mink and cause mortality (Molenaar et al., 2020). However, the spectrum of susceptible animals to SARS-CoV-2 is still unclear.
Susceptible animals to SARS-CoV-2 can be screened through animal experiments or computational simulation methods. Experimentally, the susceptible animals were evaluated through inoculation of SARS-CoV-2 virus in biosafety level 3 lab (BSL-3) (Barry Hassan et al., 2020;Rockx et al., 2020;Schlottau et al., 2020;Shi et al., 2020), which is expensive and labor-intensive. Thus, it is urgently needed to develop computational tools for prioritizing susceptible animals of SARS-CoV-2.
As we know, receptors are the biological basis for viruses to attach to and enter host cells. SARS-CoV-2 shares a high identity with SARS-CoV-1 in the receptor-binding domain of spike protein, and their receptors are all angiotensin-converting enzyme 2 (ACE2) . Thus, it is feasible to identify susceptible animals of SARS-CoV-2 by computational methods utilizing the sequence and structure alignment of ACE2 proteins across animal species and the molecular docking between ACE2 protein and the spike protein of SARS-CoV-2. The sequence or structure alignment-based methods compared the sequence or structure of ACE2 protein across the different hosts. The hosts with similar ACE2 receptors (to humans or other confirmed infected species) will have a high chance of being infected by SARS-CoV-2 (Hayashi et al., 2020;Qiu et al., 2020). Protein docking-based methods directly predict the interaction between host receptor protein and the spike protein of SARS-CoV-2. The protein docking algorithms are divided into rigid and flexible ones (Sable and Jois, 2015;Lensink et al., 2016;Xu et al., 2018;Wen et al., 2019). Rigid docking methods like ZDOCK (Pierce et al., 2014) and MEGADOCK (Ohue et al., 2014) are usually simple and less time-consuming. However, their prediction reliability is also poor. On the contrary, the flexible docking methods can provide accurate prediction at the cost of high computational resources. Commonly used flexible docking algorithms include Rosetta (Wang et al., 2007), AutoDock (Morris et al., 2009), andHADDOCK (van Zundert et al., 2016). The protein docking method can not only be applied in the research of potential host search, but also in other research fields of SARS-CoV-2, such as predicting antiviral drug candidates (Dai et al., 2020), identifying SARS-CoV-2 inhibitors (Das et al., 2020;Ton and Gentile, 2020), and manifesting the molecular mechanism of SARS-CoV-2 invasion (He et al., 2020).
This study presented a novel computational framework for systematically tracing susceptible animals to SARS-CoV-2. The binding affinities of different species to the spike protein of SARS-CoV-2 were first calculated. Then, the relationship between the evolution of human ACE2 protein and the binding affinity of SARS-CoV-2 spike protein was analyzed. Finally, the influences of a few mutations at receptor binding sites of SARS-CoV-2 on its binding affinity to ACE2 proteins across different species were simulated.

A Bioinformatics Framework for Systematically Tracing Susceptible Animals to SARS-CoV-2
We proposed a novel computational framework to trace susceptible animals to SARS-CoV-2 (Figure 1). First, we aligned the amino acids sequence of the M2 region of hACE2 protein against the NCBI non-redundant protein sequences (nr) database. Based on predefined filter rules, we identified 31 species to model the structure of their ACE2 proteins and calculated the binding affinities between their ACE2 proteins and spike protein for five SARS-CoV-2 variants, respectively. It was reported that three species, ferret, cat, and tiger, were experimentally or naturally infected with SARS- CoV-2 (McAloose et al., 2020;Shi et al., 2020;Zhang et al., 2020). Thus, we used the docking scores of the three species as the threshold values to determine susceptible animals to SARS-CoV-2. In addition, we analyzed the binding sites of a few representative species to explain how spike proteins interact with their ACE2 proteins. Finally, we simulated some mutations on human ACE2 protein and constructed the FIGURE 1 | A bioinformatics framework for systematically tracing susceptible animals to SARS-CoV-2. First, we aligned the amino acids sequence of the M2 region of hACE2 protein against the NCBI nr database and selected 31 species for structural modeling and binding their ACE2 protein with SARS-CoV-2 spike protein. The binding scores of ferret, cat, and tiger were setting as a threshold to find susceptible animals to SARS-CoV-2. Second, we analyzed the binding sites of a few representative species to explain how the spike proteins interact with their ACE2 proteins. Finally, we simulated some mutations on human ACE2 protein and viral spike protein and calculated the binding affinity between mutated/un-mutated ACE2 proteins and mutated/un-mutated viral spike proteins.
mutational human ACE2 protein model. The binding affinity prediction was performed on the mutated proteins.
The Amino Acid Sequence of the Angiotensin-Converting Enzyme 2 M2 Region of Different Species Is similar, but That of the Angiotensin-Converting Enzyme 2 Gene Is Quite Different Yan et al. (2020) reported that SARS-CoV-2 spike protein direct bonds to an M2 region of hACE2 protein. Thus, we extracted the amino acid sequence of the M2 region of hACE2 and aligned it with the NCBI non-redundant protein sequences (nr) database. There are 407 species with alignment identity greater than or equal to 70% (Supplementary Data Sheet 1), which all belonged to the Chordata phylum and contained 326 genera.
We downloaded the ACE2 protein sequences of the 407 species from NCBI (Supplementary Data Sheet 2) and aligned the amino acids sequences of the M2 region of their ACE2 proteins. By setting tiger, cat, domestic ferret as thresholds and a few other criteria (see section "Materials and Methods"), we selected 30 species plus human (overall 31 species) for subsequent analysis (Supplementary Data Sheet 3). A phylogenetic tree was constructed based on the ACE2 proteins of these species FIGURE 2 | Phylogenetic tree of 31 species based on their ACE2 protein sequences. The branch length represents the number of substitutions per site, with the positions containing gaps and missing data being eliminated. The tree was clustered into 4 main orders, namely Primates, Rodentia, Artiodactyla, and Carnivora, respectively. Primates were marked in the red frame; Rodentia was in the blue frame; Artiodactyla was in the green frame, and Carnivora was in the purple frame.
using MEGA7 (Figure 2). The phylogenetic tree showed that these species clustered four main orders: Primates, Rodentia, Artiodactyla, and Carnivora. The amino acid sequence of Primate ACE2 protein is the closest to that of humans. The suspected host bat (Rhinolophus sinicus) and pangolin (Manis javanica) were far away from the human, indicating that there might be some intermediate host if they are a genuine original host.

The Structure and Sequence Similarities Between the M2 Region of Angiotensin-Converting Enzyme 2 Protein in Humans and That in Other 30 Species Are Not Necessarily Consistent
We selected the amino acid sequences of the M2 region within ACE2 protein across 30 species (Supplementary Data Sheet 4) to predict their protein structures using the I-TASSER pipeline. We calculated the sequence and structural similarity of human ACE2 protein with that of the 30 species (Table 1). We performed a Spearman correlation test between the two vectors to test the consistency between sequence and structural similarity. The sequence and structure similarity metrics (including TM score, GDT-TS score, and RMSD) have a correlation of 0.379, 0.406, and −0.407 with a p-value of 0.039, 0.026, and 0.026, respectively, indicating that the two vectors are correlated thus statistically consistent. The top three species with similar structures to human ACE2 were Vulpes vulpes (TM score: 0.9831), Pan paniscus (TM score: 0.983), and Ursus arctos horribilis (TM score: 0.9828). However, the top three species ranked by sequence similarity are Gorilla gorilla gorilla (99.48%), Pan paniscus (99.48%), and Pan troglodytes (99.48%). Interestingly, Rhinolophus sinicus only has a sequence identity of 80.28% with the human in the M2 region of ACE2 protein, which ranks the last in the 30 species. However, its TM score is 0.9529, ranking 15th. These results indicated that variations in more amino acids in the protein sequence do not necessarily lead to more remarkable changes in structure.

SARS-CoV-2 Might Infect a Few Species in Close Contact With Humans
The HADDOCK score estimates the stability of the complex of ACE2 protein binding with viral spike protein. A complex with HADDOCK scores less than 0 is considered stable. We showed the HADDOCK scores of the ACE2 protein from 31 studied species docking with spike protein in SARS-CoV-2 (Wuhan) and Omicron in Table 2. Equus caballus ranked first in the list, followed by Oryctolagus cuniculus for SARS-CoV-2 (Wuhan). In contrast, Panthera tigris altaica ranked first, followed by Mus musculus for Omicron. Previous studies have suggested that SARS-CoV-2 could infect Homo sapiens, Panthera tigris altaica, Mustela putorius furo, Felis catus and Rhinolophus sinicus rank 20, 10, 15, 5, and 6, respectively in the list of Wuhan. Thus, we suspect that the top 20 species are potentially susceptible animals for SARS-CoV-2 Wuhan. The 20 species include wild animals, husbandry animals, and pets, indicating that the infection of SARS-CoV-2 is unbiased across these three categories of animals.
There is no significant correlation between ACE2 sequence similarity and ACE2-RBD structural similarity. The similarity of M2(ACE2)-RBD(SARS-CoV-2) structure between humans and other species was evaluated using RMSD and TM score, and the result is shown in Table 3. Surprisedly, compared with M2(ACE2)-RBD(SARS-CoV-2 Wuhan) structural similarity between human beings with other 31 species, M2(ACE2)-RBD(Omicron) decreased for most of the 31species.  Table 2). In addition, a phylogenetic tree for 7 SARS-CoV-2 variants was constructed with spike protein sequences (Figure 3), showing an intriguing evolutionary relationship between Omicron with other variants that evolved in human patients. We speculated that the progenitor of Omicron rapidly evolved by accumulation of mutations conductive to infecting that host and then jumped into human beings. Zhou et al. (2020) reported a cryo-electron microscopy structure of the complex, in which human ACE2 protein binds with the receptor-binding domain (RBD) of the viral spike protein. The structure indicated that human ACE2 protein interfaced with the RBD of viral spike protein by seven residues: Q24, D30, H34, Y41, Q42, K353, and R357 of ACE2 interact with Q474, K417, Y453, N501, Q498, N501, and T500 of RBD.
At the N terminus of cat ACE2 protein (Figure 4A), we found that the ACE2 protein binds with RBD by three H-bonds. The THR739, TYR780, and ASN770 of cat ACE2 interacted with ASP428, ASP389, and LEU518 of RBD ( Figure 4B). These H-bonds were less than 3 Å in the distance and provided a strong binding force.
The HADDOCK score of Mustela putorius furo was larger than that of Felis catus, and its ACE2 protein had six H-bonds with spike protein (Figure 4C) Molecular docking between 31 ACE2 proteins and RBD of the Spike protein of SARS-CoV-2 were conducted for five strains (including Wuhan, Gamma, Delta, and Omicron), respectively. Of 31 species, 20 species exhibited the lowest value of HADDOCK score of ACE2 docking with Spike protein of Omicron and nine species for Delta variants (Figure 5), indicating that binding affinity of spike protein with ACE2 of host elevated with the accumulation of mutations in SARS-CoV-2 variants. For the Delta strain, the species with the strongest binding affinity to the Spike protein was Macaca mulatta. For the Omicron strain, the strongest binding affinity is Panthera tigris altaica.
The structural similarity of M2(ACE2)-RBD(SARS-CoV-2) between humans and other species were compared by the TMalign using metrics TM score and RMSD, and the results were shown in Supplementary Figures 1, 2, respectively. Surprisingly, compared with M2(ACE2)-RBD(SARS-CoV-2) structural similarity in the wild-type Wuhan strain, the similarity decreased in variants for most species of 31 animals (Supplementary Figures 1, 2). However, the most striking finding to emerge from our study was that structural similarity of M2(ACE2)-RBD(ARS-CoV-2) between Homo sapients and Octodon degus and was elevated for Delta virus compared with that of Wuhan strain. Furthermore, the structural similarity of M2(ACE2)-RBD(ARS-CoV-2) between Homo sapients and Bos Bold characters represent species with increased binding affinity for ACE2 with SARS-Cov-2 Omicron, and numbers in bold indicate altered binding affinity.
taurus was elevated for the Omicron virus compared with that of the Wuhan strain.

Human Angiotensin-Converting Enzyme 2 Protein Q340 Mutation Improves the Binding Affinity Between hACE2 and SARS-CoV-2
To test the effect of mutations of ACE2 on the binding affinity between hACE2 and SARS-CoV-2, we chose 11 residues to mutate and modeled the protein structure of the mutated ACE2 proteins. These residues were selected based on the frequency of mutations in other species (compared to humans). Table 4 shows the result of a few mutational ACE2 proteins docking with spike protein. The HADDOCK score of the original human ACE2 protein was −100.6 ± 17.5, which is less than the HADDOCK score of these mutated ACE2 proteins. This result indicates that the mutant ACE2 protein has a stronger binding capacity to the spike protein. Especially, the mutation Q340R has the lowest HADDOCK score of -139.2, which is the most stable in binding. It is known that residue 340 is not a binding site, suggesting that mutations in the non-binding sites can also affect the binding of ACE2 protein and spike protein. In the virus infection process from wild hosts to humans, SARS-CoV-2 may gradually adapt to the human ACE2 protein.

Blast Search on Species With Sequences Similar to Human Angiotensin-Converting Enzyme 2 Gene and the M2 Region of Human Angiotensin-Converting Enzyme 2 Protein
The amino acids (AA) sequence of the M2 region of hACE2 (1R42) was downloaded from the PDB database (PDBid: 1R42). The amino acid sequence was aligned with the NCBI nr database by blastp (Shiryev et al., 2007;Camacho et al., 2009). BLAST parameters were chosen as follows: e-value was set to 10; the The bold number indicates that Bos taurus M(ACE2)-RBD(Omicron) has the highest structural similarity to Homo sapiens. cost of gap-open and gap-extend was set to 1; the maximum number of alignment results was set to 1E8. BLAST results with an e-value more than 0.01 and a similarity of less than 70% were filtered out.

Selecting Candidate Species
Thirty-one species, including humans and 30 other species, were selected for further analysis according to four rules. Firstly, the similarity between the M2 region of human ACE2 protein and the given species was calculated; a species more similar than Rhinolophus sinicus was selected. We selected Rhinolophus sinicus as a borderline species since its infected coronavirus has 96% similhasy to SARS-CoV-2 at the whole-genome level, and it is not quite similar to humans. Secondly, six animals with phylogenetically identical to the human ACE2 gene were selected, including Chlorocebus sabaeus, Gorilla gorilla gorilla, Macaca mulatta, Pan paniscus, Pan troglodytes, and Pongo abelii. Thirdly, four animals, including Felis catus, Mustela putorius furo, Panthera tigris altaica, and Manis javanica, were selected since they are reported to be infected with SARS-CoV-2 or coronavirus  with high similarity to SARS-CoV-2 (Shi et al., 2020). The selected 31 animal species cover wild animals, pet, and husbandry animals.

Construction of Phylogenetic Tree
The ACE2 protein sequences of the 31 selected species were aligned by ClusterX (Thompson et al., 2002). Phylogenetic analysis was conducted using the maximum likelihood method based on the JTT matrix-based model (Jones et al., 1992). One thousand bootstrap replicates were used to calculate node support by MEGA7 (Kumar et al., 2016). The phylogenetic tree was displayed and annotated by the online tool iTOL (Letunic and Bork, 2019). We constructed the phylogenetic tree for S proteins of 7 SARS-CoV-2 variants, including Wuhan, alpha, beta, gamma, kappa, delta, and Omicron, using the same approach as the ACE2 phylogenetic tree. Except for Omicron, the Spike protein sequences were retrieved from protein data bank (PDB) for six SARS-CoV-2 variants. The genome sequence for Omicron was downloaded from the GISAID database (Shu and McCauley, 2017), and the S protein sequence was annotated by local BLAST 2 . Modeling Angiotensin-Converting Enzyme 2 Protein Structure The ACE2 protein structures of different species were modeled by the I-TASSR analysis, which consists of three steps, including (1) threading, (2) structural assembly, and (3) model selection and refinement (Roy et al., 2010;Yang et al., 2015). In the first step, LOMETS threaded the query protein sequence using a library of non-redundant structures to identify structural templates. In the second step, the topology of a full-length model was constructed by reassembling the continuously aligned fragments excised from templates. The structure of unaligned regions was built from scratch by ab initio folding. In the third step, the simulation of fragment assembly was utilized again as the starting selected cluster centroids. The final structural model was generated by building all-atom models from Cα traces using the optimized hydrogen-bonding networks. The similarity of human ACE2 protein structure and 30 species' was calculated by TMscore program implemented in I-TASSER software using metrics TM score, GDT-TS score, and RMSD (Yang et al., 2015).

Molecular Docking Between Angiotensin-Converting Enzyme 2 Protein and SARS-CoV-2 Spike Protein
The docking score of the HADDOCK server (van Zundert et al., 2016) was used to predict the binding affinity between SARS-CoV-2 spike protein and the ACE2 protein of a particular species. A few hACE2 residues, including Q24, D30, H34, Y41, Q42, K353, and R357, were set as the activated sites of the ACE2 receptor in HADDOCK since these residues were reported to effectively bind to the viral spike protein . In addition, the activated sites of spike protein included K417, Y453, Q474, Q498, T500, and N501, respectively. Molecular docking between 31 ACE2 proteins and spike protein of five SARS-CoV-2 were conducted, respectively. The structural similarity of M2(ACE2)-RBD(SARS-CoV-2) between humans and other species were compared by the TMalign program implemented in I-TASSER software using metrics TM score and RMSD (Yang et al., 2015).

Modeling the Mutation of hACE2 Protein
To predict the potential effect of mutants in hACE2, we mutate a few residues in hACE2, which have single nucleotide polymorphism (SNP) in 50% of species other than humans. Then, the Python library MODELER was used to model these mutated hACE2 proteins. Again, HADDOCK (van Zundert et al., 2016) was used to predict the binding affinity between the mutated/unmutated hACE2 proteins.

DISCUSSION
The SARS-CoV-2 pandemic has already resulted in more than one hundred million human cases and caused a substantial economic loss. However, a detailed spectrum of susceptible animals to this virus is still unclear. In this study, we predicted susceptible animals to SARS-CoV-2 through a bioinformatics framework. Besides humans, we found that 22 species, including primate, pet, husbandry, and wild animals, were potentially susceptible to SARS-CoV-2, including cat, ferret, and tiger, which have already been reported to be naturally or experimentally infected with SARS-CoV-2.
We found some inconsistency among the amino acids sequence similarity of ACE2, the structural similarity of ACE2, and the binding affinity between ACE2 protein and SARS-CoV-2 spike protein across different species. The sequence similarity shows the evolutionary relationship among other species, while the structural similarity represents the similarity of protein folding (Zhang and Skolnick, 2005). Since the influence of each protein site is different, which might be possible that close evolutionary species exhibit quite different protein structures when important mutations occur (Zhang and Skolnick, 2005). Similarly, the inconsistency between structure similarity and binding affinity might be caused by the fact that the binding between ACE2 protein and viral spike protein only happens at active binding sites and the importance of each site is different. For example, Canis lupus familiaris (dog) has been reported to be infected by SARS-CoV-2 (Sit et al., 2020). The structural comparison showed that the ACE2 protein of dogs is quite different from that of humans. However, the docking results illustrated that ACE2 of dog can bind to the S protein of SARS-CoV-2. Therefore, we believe that protein-docking analysis is more reliable in predicting SARS-CoV-2 infection than sequence and structural similarity.
Though a few species, such as Bos Taurus have high docking scores, studies are suggesting that these species may not be infected by SARS-CoV-2 (Schlottau et al., 2020;Shi et al., 2020). Similar to SARS-CoV and MERS-CoV, the SARS-CoV-2 spike protein (S) has S1 and S2 (Li, 2016;Qing et al., 2020), with the S1 subunit containing the RBD region. Bloise et al. (2020) found that the infection of SARS-CoV-2 depends on both ACE2 and TMPRSS2 of host cells (Matsuyama et al., 2020). Specifically, after the S1 subunit of spike protein binds with ACE2 protein, the host TMPRSS2 protein cleavages the S protein into S1 and S2 subunits. The S2 subunit makes virus fusion with the host cell. We searched TMPRSS2 in the NCBI database and found that some species did not contain the TMPRSS2 gene, which might be why some species could not be infected even though their ACE2 has high docking scores with the S protein of SARS-CoV-2.
Our findings illustrated that the susceptibilities of canine, equine, and swine are a little bit higher than that of feline. However, these findings are not consistent with a previous study, which shows that feline belongs to the medium susceptible animal group, and swine, equine and canine fall into the low susceptible animal group (Damas et al., 2020). The possible reason for this discrepancy is that they ranked susceptible animals based on 25 known binding residues of ACE2 and its structure; however, we ranked susceptible animals according to HADDOCK score for the binding affinity between S protein and host ACE2. In the present study, we found that ACE2 of Rhinolophus sinicus showed low binding affinity to the S protein of SARS-CoV-2, which is consistent with Wu et al. (2020). In addition to Wu's study, we also illustrated that Heterocephalus glaber, Mesocricetus auratus, Chinchilla lanigera, and Ursus arctos horribilis are susceptible animals to SARS-CoV-2. However, these prediction results should be experimentally confirmed in the future.
Biological methods screened susceptible animals through virus infection in vivo or pseudo-virus in vitro. However, they usually require labs with high biosafety levels and are time and cost-intensive. In addition, it is hard to catch wild animals and construct animal models (Bao et al., 2020;Bloise et al., 2020;Gand et al., 2020;Gorshkov et al., 2020;Hassan et al., 2020;Hou et al., 2020;Preziuso, 2020;Wang et al., 2020). Compared to computing methods, the advantage of biological methods is more accurate. In contrast, computing methods have advantages such as being fast, cheap, safe, and could predict wild animals. SARS-CoV-2 infection is a complex process (Hussain et al., 2020;Korber et al., 2020;Walls et al., 2020;Yan et al., 2020), which involves the interaction between viruses and hosts. Prevalent computing methods often consider one or two biological processing, such as binding and fusion, to predict the interaction between virus and host. Sometimes, a few results are inconsistent with biological experiments. For instance, Erinaceus europaeus was predicted to be more susceptible to SARS-CoV-2 than that of feline in our study; however, Wu's research proved that pseudotyped SARS-CoV-2 fails to efficiently transduce into cells expressing ACE2 of European hedgehog, lesser hedgehog tenrec . Therefore, bioinformatics results need to be validated by biological assays.

CONCLUSION
We illustrated that 23 animal species are potentially susceptible to the SARS-CoV-2 virus, including primates, companion pets, husbandry animals, and other wild animals, through a bioinformatics framework. These findings provide novel insight into tracing SARS-CoV-2, identifying susceptible animals, and controlling and preventing the SARS-CoV-2 pandemic.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
ML, AW, and JY designed the study. AW retrieved the data. AW and LW performed the data analysis. HS and AW wrote the manuscript. BW and GT reviewed the manuscript. All authors contributed to the article and approved the submitted version.